Production of MarathonRT and its comparison with commercial reverse transcriptases for tRNA sequencing library preparation

As tRNA research gains momentum, several high-throughput sequencing methods have emerged for quantitative analysis of tRNA isoacceptors in cells. However, the strong secondary structure and rich post-transcriptional modification of most tRNA molecules pose significant challenges for reverse transcriptases, thus hampering library preparation and introducing quantification biases.

Current approaches rely on processive next generation reverse transcriptases (ngRTs) – they successfully overcome these problems, albeit with the potential caveat of high experimental costs. Here, we introduce a recombinant MarathonRT (MRT) protein with a C-terminal chitin binding domain (CBD), for which we present a simple and robust one-step purification protocol that yields over 18,000 enzymatic reactions per 0.5 L of expression culture.